Year 2020, Volume 7 , Issue 1, Pages 211 - 220 2020-06-28

The Transfection of HepG2 cells with Truncated β-Catenin Coding Expression Vector
HepG2 Hücrelerinin Kısa Uçlu β-catenin Kodlayan İfade Vektörüyle Transfeksiyonu

Oğuzhan KARAOSMANOĞLU [1]


β-catenin is an effector protein in Wnt signaling. β-catenin mutations are reported in the development of many diseases such as autism, colorectal carcinoma, developmental delay, intellectual disability, neurodegeneration, skin, hair and facial anomalies. Exon 3 deletion mediated truncations of the β-catenin associated with these diseases. Therefore understanding the functions of wild type and exon 3 deleted forms of β-catenin may provide an enhancement in the treatment of many diseases. However, to conduct controlled experiments, there could be a demand for the expression vectors that code for wild type and exon 3 deleted forms of β-catenin and originated from the same organism. Since it has long been known that HepG2 cells are heterozygous for β-catenin, in this study, it was found worthy of constructing the expression vectors from the total RNA of HepG2 cells. Then the utility of truncated β-catenin coding pcDNA3.1/CTNNB1 expression vector for upregulation of truncated β-catenin in HepG2 cells was examined. To this end, RNA was isolated from HepG2 cells, cDNA fragments were amplified by polymerase chain reaction (PCR), expression vectors were constructed then sequenced from 5’-prime regions. Following the BLAST analysis, it was concluded that both truncated and wild type β-catenin coding pcDNA3.1/CTNNB1 expression vectors were successfully cloned in E. coli cells. Interestingly, when the parental HepG2 cells were transfected with exon 3 deleted expression vector, β-catenin protein levels were not affected. Moreover, cellular morphology and population doubling time were not significantly altered.
β-katenin, Wnt sinyalleşmesinde bir efektör proteinidir. β-katenin mutasyonları; otizm, kolon kanseri, gelişimsel gecikme, zihinsel engel, nörodejenerasyon, baş, deri, ve yüz anomalileri gibi çok sayıda hastalıkların gelişmesinde raporlanmıştır. Bu hastalıklar, özellikle ekson 3 delesyonu aracılı β-katenin kısalmalarıyla ilişkilendirilmiştir. Bu nedenle β-katenin proteininin yabani tip ve ekson 3 delesyonlu formlarının fonksiyonlarını anlamak çok sayıda hastalığın tedavisinde ilerlemeyi sağlayabilecektir. Kontrollü deneyler kurmak için, yabani tip ve ekson 3 delesyonlu β-catenin formlarını kodlayan ve aynı organizmadan kökenlenen ekspresyon vektörlerine ihtiyaç duyulabilmektedir. HepG2 hücrelerinin β-katenin proteinleri bakımından heterozigot olduğu uzun zamandır bilindiği için, bu çalışmada, yabani tip ve ekson 3 delesyonlu ekspresyon vektörlerini HepG2 hücrelerinin toplam RNA'sından oluşturmanın değerli olabileceği düşünülmüştür. Bunun için, HepG2 hücrelerinden RNA izole edilmiştir, cDNA parçaları polimeraz zincir reaksiyonu (PZR) ile çoğaltılmıştır, ifade vektörleri oluşturularak 5’-uçlarından dizilenmiştir. BLAST analizi sonrası hem ekson 3 delesyonlu hem de yabani tip β-katenin kodlayan pcDNA3.1/CTNNB1 ifade vektörlerinin E. coli hücrelerine başarıyla klonlandığı sonucuna varılmıştır. İlginç bir şekilde, HepG2 hücreleri ekson 3 delesyonlu ifade vektörü ile transfekte edildiğinde, β-katenin protein seviyesi etkilenmemiştir. Dahası hücre morfolojisi ve populasyon ikilenme zamanı anlamlı ölçüde değişmemiştir.
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Primary Language en
Subjects Basic Sciences
Journal Section Articles
Authors

Orcid: 0000-0003-2028-7339
Author: Oğuzhan KARAOSMANOĞLU (Primary Author)
Institution: KARAMANOGLU MEHMETBEY UNIVERSITY, KAMİL ÖZDAĞ FACULTY OF SCIENCE
Country: Turkey


Supporting Institution TUBITAK
Project Number 114S207
Thanks The author is grateful to The Scientific and Technological Research Council of Turkey (TUBITAK) for supporting this research with the grant number of 114S207.
Dates

Application Date : December 12, 2019
Acceptance Date : May 16, 2020
Publication Date : June 28, 2020

APA Karaosmanoğlu, O . (2020). The Transfection of HepG2 cells with Truncated β-Catenin Coding Expression Vector . Bilecik Şeyh Edebali Üniversitesi Fen Bilimleri Dergisi , 7 (1) , 211-220 . DOI: 10.35193/bseufbd.658677